Kappa opioid receptor (KOR) is one of the three opioid receptors (, ? and ?). The receptor belongs to the rhodopsin sub- family of the G protein-coupled receptors. Activation of the KOR in vivo produces many effects, including analgesia, antipruritic effects, water diuresis, dysphoria / aversion, sedation, motor incoordination and hypothermia. KOR agonists are potentially useful as analgesics, antipruritic agents and water diuretics, whereas KOR antagonists may be useful for treatment of depression, anxiety and drug addiction. The claustrum has the highest level of KOR in the central nervous system, yet its roles in KOR-mediated behaviors have not been elucidated. The claustrum is a long thin band-like grey matter extending in the rostral-caudal direction in the ventrolateral forebrain of all therian mammals. The claustrum has extensive reciprocal connections with the cortex and, to lesser extents, with other brain regions, including the nucleus accumbens, thalamus and striatum. The function of the claustrum remains unknown despite of extensive neuroanatomical studies. We have generated a knockin mouse line expressing a fusion protein of KOR conjugated with the fluorescent protein tdTomato (KtdT). The homozygous KtdT/KtdT mice displayed similar KOR agonist-induced anti-scratching behavior and sedation as the wildtype mice. In brain sections, immunohistochemistry (IHC) using specific antibodies against the red fluorescent protein (RFP), which recognizes tdT, revealed that KtdT immunoreactivity had similar distribution as autoradiography of [3H]U69,593 binding to the KOR in wildtype mice. The KtdT/KtdT knockin mice allows visualization of the KOR at a much higher resolution than that afforded by autoradiography. The specific aims of the proposed study are as follows. For Specific Aim 1, we will examine the presence of some nonpeptide neurotransmitters and their relationship to the KOR in the claustrum. Availability of KtdT/KtdT mice makes it possible to address this question by double IHC using specific antibodies against markers of the neurotransmitters and antibodies against the RFP for KtdT. Double in situ hybridization (ISH) will be used to examine colocalization of mRNAs of KOR and neurotramitter markers in cell bodies. Neurotransmitters to be examined include glutamate, GABA, serotonin, and catecholamines, which have been demonstrated in the claustrum. For Specific Aim 2, we will generate and characterize knockin mouse lines with conditional deletion of the KOR in the claustrum. Expression of Gnb4 is highly restricted to the claustrum. The mouse line will be generated by cross-breeding a floxed KOR line with Gnb4-IRES2-Cre-D knockin mice. The resulting Gnb4KOR-/- mice will be confirmed by elimination or a great reduction of KOR in the claustrum and examined for KOR decreases in any other brain regions, which will yield information on projection from the claustrum. Gnb4KOR-/- mice will be compared with the control mice to determine the role of KOR in the claustrum in KOR-mediated behavioral responses, including analgesic, anti-scratching and sedative effects, motor incoordination and conditioned place aversion. The proposed studies will be the first to elucidate the roles of claustrum KOR in KOR-mediated behaviors and greatly enhance our understanding of in vivo KOR neurobiology. In addition, the results will shed light on functions of the claustrum, which have been one of the most enigmatic regions in the brain.